PROJECT SUMMARY A prevalent route to aneuploidy for diploid cells is the occurrence of a Whole-Genome Doubling (WGD) followed by chromosome loss. WGD buffers against deleterious effects of changes in essential genes during tumor cell evolution. However, WGD also results in a doubling of centrosome content, which can be deleterious during mitosis. This is because each of the centrosomes nucleates microtubules, resulting in a multipolar spindle. There are two potential outcomes when this happens: cells can initiate a multipolar division, which is lethal, or they can re-organize the multipolar spindle into a bipolar spindle by clustering the extra centrosomes. The latter outcome allows for bipolar cell division and survival, but with an increased rate of chromosome segregation errors. After WGD, normal untransformed human cells experience both cell division outcomes with approximately equal frequency. Cancer cell lines, by contrast, are generally more proficient at clustering extra centrosomes, but how this occurs is not known. The long-term goal of this work, therefore, is to define mechanisms that allow cells to survive mitosis after WGD. The overall objective of this proposal is to define how cancer-associated mutations and deletions in protein phosphatase 2A (PP2A) alter mitosis after WGD. The central hypothesis is that PP2A inactivation increases the viability of cells after WGD through enhanced centrosome clustering and reduction in chromosome segregation errors. This hypothesis is based on studies of a prevalent mis-sense mutation in PP2A-A, a core component of heterotrimeric PP2A complexes. The rationale for the proposed research is that knowledge of how cancer cells mitigate the stress associated with WGD, and more specifically, how recurrent PP2A-A mutations change mitosis, could potentially be used to therapeutically target those pathways in cancer cells. Our objective will be realized by the following two aims: (1) Identify how PP2A controls centrosome clustering. Phosphorylation of NuMA and TPX2, two regulators of spindle assembly, is altered by PP2A-A mutation. Complementary in vivo and in vitro approaches will be used to determine how this impacts centrosome clustering after WGD. (2) Identify how PP2A controls the fidelity of chromosome segregation. High-resolution live-cell imaging, FRET-based phosphorylation biosensors, and quantitative immunofluorescence of endogenous kinetochore substrates will be used to test the hypothesis that PP2A-A mutation reduces chromosome segregation errors through altered phospho- signaling at kinetochores. The long-term impact of PP2A-A mutation on genome stability in cells that experience WGD will also be determined. Successful completion of this work will establish how human cancers `fine-tune' phosphorylation to ensure proliferation after WGD. This knowledge could inform on strategies to target tumor cells with ploidy changes or supernumerary centrosomes. ! !